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. 2022 Jan 27;12:1443. doi: 10.1038/s41598-022-05161-7

Figure 5.

Figure 5

Overexpression of oxygen stable HIF mutant proteins does not increase small EV release in normoxic HEK293T cells. HEK293T cells were transiently transfected with plasmids coding for normoxia-stable HIF1α P402A/P564A (mHIF1α) or HIF2α P405A/P531A (mHIF2α) mutants as indicated in the methods section. (a) Immunoblot of cellular lysates. Cell homogenates were resolved in SDS-PAGE and immunoblotted with antibodies for HIF1α, HIF2α or  β-tubulin. Representative western blot analysis is shown on top and quantification of immunoblots carried out in Image J (NIH) is shown underneath. Graphs show the mean ± SEM of a representative experiment of N = 2 experiments performed each with two biological replicates. Statistical analysis: Two-way ANOVA, followed by Sidak’s multiple comparisons test. **p < 0.01, ***p < 0.001. (b) Immunolocalization of mHIF1α and mHIF2α in HEK293T cells. Following 24 h after transfection cells were fixed, permeabilized and stained with antibodies against HIF1α and HIF2α as described in the methods section. Nuclei were stained with DAPI. (scale bar = 50 µm). (c) mHIF1α and mHIF2α expressing cells show induction of mRNAs of hypoxia activated genes. Total RNA was isolated from cells transfected with mHIF1α and mHIF2α expressing plasmids. Following RNA extraction a RT-qPCR was performed to analyse the transcription levels of genes known to be highly upregulated by HIF (VEGFA and SLC2A1) and a third one known for being generally stable and not affected by HIF transcriptional program (VEGFB) as control. Graphs show the expression levels calculated as the 2−ΔΔCt values for each gene normalised to β-actin. Graphs show mean ± SEM of n = 3 replicates, each analysed in duplicate. Statistical analysis: One-way ANOVA, *** indicates p < 0.001. (d) Quantitation of small EVs by measurement of vesicles in the supernatant of the 10,000×g centrifugation of the conditioned media. Samples were of untransfected cells or from cells overexpressing either mHIF1α or mHIF2α. Data was normalized to the amount of total protein content present in the cellular lysate. Graph shows the mean ± SEM of a representative experiment of N = 2 experiments performed each with two biological replicates. (e) Immunoblot of cells expressing both mHIF1α and mHIF2α. Cellular lysates obtained from untransfected cells or cells expressing both mHIF1α and mHIF2α were resolved by SDS-PAGE and immunoblotted with antibodies againts HIF1α, HIF2α or β-tubulin. A representative western blot analysis is shown on the left. The graphs on the right show the quantification data (mean ± SEM) of N = 4 experiments each performed in duplicate. Statistical analysis: Two-way ANOVA, followed by Sidak’s multiple comparisons test. * p < 0.05. (f) Quantitation of small EVs by measurement of vesicles in the supernatant of the 10,000×g centrifugation of the conditioned media. Samples were of untransfected cells or from cells overexpressing mHIF1α and mHIF2α. Data was normalized to the amount of total protein content in the cellular lysate. Graph shows the mean ± SEM of N = 4 experiments performed each in duplicate.